More than 20% of the population suffer from type I allergic reactions. The prevalence of asthma, hay fever and other IgE-mediated diseases has increased dramatically in industrialized countries over the last decades, making allergies a very serious public health problem (Beasley et al., J Allergy Clin Immunol 105: 466-472, 2000).
For example, asthma prevalence rates increased by 75% in the United States between 1980 and 1994 (Doyle, Sci Am 282: 30, 2000).
The most important predisposing factor for the development of allergic diseases is atopy, the genetic predisposition to produce allergen-specific IgE. Childhood asthma is mainly found in patients who are atopic and sensitized to common environmental allergens including house dust mites, tree and grass pollens. Recent epidemiological studies suggest a close correlation between serum IgE levels and the presence or severity of asthma (Burrows et al., N Engl J Med 320: 271-277, 1989). In contrast, patients with a non-allergic asthma have negative skin tests to common allergens and normal total Ige serum levels. The association of increased IgE serum levels is also obvious for other allergic diseases such as seasonal allergic rhinitis, insect venom allergy or food allergies.
Symptoms of type I allergic reactions are due to the release of mediators (e.g. histamine) resulting from allergen-mediated crosslinking of IgE (immunoglobulin E) antibodies bound to IgE receptors on effector cells. The high-affinity IgE receptor FcεRI is mainly expressed on mast cells and basophils (Ravetch and Kinet, Ann Rev Immunol 9: 457, 1991), whereas the low-affinity IgE receptor FcεRII (CD23) is expressed on B cells (Conrad, Ann Rev Immunol 8: 623-645, 1990). Free serum IgE has a very short half-life of about two and a half days, while mast cells remain sensitized for up to 12 weeks following binding of IgE to high-affinity receptors.
Treatment modalities of type I allergies have substantially improved over the last decades. Allergen-specific immunotherapy (SIT) represents a curative approach. A rise in allergen-blocking IgG antibodies, particularly of the IgG4 class (Reid et al., J. Allergy Clin. Immunol. 78: 590-600, 1986), a reduction in the number of mast cells and eosinophils, and a decreased release of mediators (Varney et al., J. Clin. Invest. 92: 644-651, 1993) were found to be associated with successful SIT. However, for some patients including polysensitized and very young patients SIT is not applicable. The most advanced treatment modality that targets the pathophysiological cascade of allergen-mediated immune reactions earlier and in a more general way than SIT, is the inhibition of IgE responses by anti-IgE antibodies. The binding site of IgE for the high-affinity IgE receptor FcεRI is located within the third domain of the heavy chain, Cε3. A murine antibody, MAE1, was generated that recognizes the same residues in the Cε3 domain of IgE that are responsible for binding to FcεRI (Saban et al., J Allergy Clin Immunol 94: 836-843, 1994). To avoid sensitization to the murine antibody, a humanized version, containing 95% of a human IgG1 antibody and only 5% of the murine antibody, was constructed and named recombinant humanized monoclonal antibody (rhuMAb)-E25 (Presta et al., J Immunol 151: 2623-2632, 1993) or omalizumab (Xolair®). The main features of this anti-IgE antibody include a) recognition and binding to serum IgE, but not to IgG or IgA, b) inhibition of IgE binding to FcεRI, c) no binding to IgE bound to mast cells or basophils, thereby avoiding degranulation (‘non-anaphylactic antibody’), and d) its capability to block mast cell degranulation upon passive sensitization in vitro or challenge with allergen in vivo. Treatment with omalizumab (Xolair®) also reduces the number of FcεRI receptors on basophils in atopic patients. Since omalizumab (Xolair®) binds to any IgE molecule irrespective of its allergen specificity, this therapeutic approach provides a valuable alternative to SIT. Recent clinical trial have demonstrated that anti-IgE is an effective agent for the therapy of allergies including moderate to severe allergic asthma and seasonal allergic rhinitis (Hamelmann et al., Allergy 57: 983-994, 2002; Hamelmann et al., Curr Opin Allergy Clin Immunol 3: 501-510, 2003).
In order to assess the success of anti-IgE treatment, methods for the in vitro measurement of IgE antibodies are required that allow differentiation between complexed and non-complexed serum IgE.
Currently available methods for the in vitro determination of serum IgE include the radio-allergosorbent test (RAST), various enzyme-linked immunosorbent assays (ELISA) and other IgE-binding techniques such as immunoelectrophoresis, immunoblot and immunodotblotting. RAST and ELISA assays are performed in three steps including immobilization of the standard/reference allergens on a solid phase (e.g., the well of a microtiter plate), binding of the IgE antibodies in the serum of a patient to the immobilized allergens, and determination of coupled IgE antibodies by labelled anti-IgE antibodies. All of these techniques, however, utilize for the detection of bound IgE polyclonal or monoclonal anti-IgE antibodies that are not capable of differentiating between complexed and non-complexed serum IgE. Since these antibodies recognize other epitopes than those residues in the Cε3 domain of IgE that are responsible for binding to FcεRI, free IgE as well as anti-IgE/IgE complexes are detected.
A new strategy for detecting allergen-specific antibodies in serum is the bead array technology. These multiplex assays can be performed in the flow cytometer or in any other similar analytical equipment that allows for the discrimination of different particles on the basis of size and color. The bead array technology employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soluble analytes from a single serum, plasma, or tissue fluid sample. The analytes can also be allergen-specific antibodies of the IgE subclass. For example, for the detection of allergen-specific antibodies specific capture beads carrying immobilized standard/reference allergens, are mixed with phycoerythrin-conjugated detection antibodies and are then incubated with test samples to form sandwich complexes. However, the polyclonal or monoclonal anti-IgE capture antibodies utilized for this technique are not suitable for the determination of non-complexed IgE due to the lack of specificity for the residues in the Cε3 domain of IgE that are responsible for binding to FcεRI.
In addition to solid-phase technology, fluid-phase systems have been established for the in vitro measurement of allergen-specific antibodies in serum. In these immunoassays, modified allergens are employed. The modified allergens contain one or more residues (e.g., biotin) that allow for subsequent binding of allergen-serum antibody complexes to a solid phase coated with a corresponding binding protein (e.g., streptavidin). The soluble polymer/copolymer support systems utilized in these assays, increase the number of binding sites and, thereby, the detection sensitivity. The most advanced fluid-phase assays for the detection of allergen-specific IgE antibodies utilize an enzyme-enhanced chemiluminescent enzyme immunoassay technique for the quantification of complexed specific antibodies. For example, for the detection of allergen-specific IgE antibodies, streptavidin-coated beads, biotinylated liquid allergens, and the patient's sample are incubated, and after a spin wash coupled IgE antibodies are detected with an alkaline phosphatase-labelled monoclonal anti-human IgE antibody using a chemiluminescent substrate (e.g., phosphate ester of adamantyl dioxetane). Again, the labelled monoclonal anti-human IgE antibody utilized for these fluid-phase systems detects free IgE as well as anti-IgE/IgE complexes.
One possibility to overcome these problems is the use of monoclonal antibodies with specificity for the residues in the Cε3 domain of IgE that are responsible for binding to FcεRI such as murine monoclonal antibody MAE1 or humanized monoclonal antibody E25 (omalizumab, Xolair®) as capture and/or detection antibody for the above listed IgE quantification procedures. However, the use of antibodies of mammalian origin raises a number of additional difficulties in practice due to the presence of rheumatoid factor (RF) and human anti-mouse IgG antibodies (HAMA) in serum samples. RF and HAMA are probably the most well known causes of false positive or false negative reactions in immunological assays (Boscato L M, Stuart M C, Clin Chem 34, 27-33, 1988). RF is an auto-antibody that reacts with the Fc part of IgG.
The disease most often associated with RF is rheumatoid arthritis, but RF can be found in serum from patients with many other diseases and also in 3-5% of healthy blood donors (Johnson P M, Faulk W P, Clin Immunol Immunopathol 6, 414-430, 1976). Production of HAMA is mainly the result of therapeutic approaches with mouse monoclonal antibodies, but HAMA may also be found in serum from patients who have not been treated with antibodies. RF or HAMA may react with both the capture antibody and the detection antibody in a sandwich assay, thereby mimicking antigen activity. A reaction with the detection antibody results in formation of an immune complex which may influence the activity of the detection antibody. HAMA may also react with the antigen-binding region of the detection antibody, thereby impairing or inhibiting antigen binding. The problem of RF and HAMA interference will increase as the sensitivity of the assay increases. Interference by anti-IgG antibodies and antibody-binding substances have been demonstrated in approximately 40% of serum samples from healthy individuals in an immunoradiometric assay (Boscato L, Stuart M, Clin Chem 32, 1491-1495; 1986). The prevalence of human anti-mammalian antibodies causing potential interferences in immunological assays varies from 1-80% in the general population (Kricka L J, Clin. Chem. 45, 942-956, 1999).
Furthermore, some mammalian IgG antibodies bound to a solid phase as well as antigen-antibody complexes comprising such antibodies, can activate the human complement system (Larsson A, Sjoquist J, J Immunol Methods 119, 103-109, 1989). Activated C4 molecules bind to the Fab region of IgG and may interfere with the antigen binding (Campbell R D, et al., Biochem J 189, 67-80, 1980). In clinical laboratories, most analyses are performed on serum samples. A newly obtained serum sample contains active complement, but the activity declines during storage and handling. Accordingly, the complement activity may vary between different patients and also between different samples from the same patient. To avoid activation of the complement cascade, EDTA is often included in tubes used for blood sampling.
EDTA prevents complement activation and coagulation by sequestering calcium ions. Most of the standards and controls used have been stored and contain an inactive complement system. This difference in activity between the samples and the standards will cause erroneous results. Complement activation was shown to interfere in an immunometric TSH assay and depressed the TSH values by up to 40% (Kapyaho K, et al., Scand J Clin Lab Invest 49, 211215, 1989).
In principle, the above mentioned problems, e.g. cross-reactivity and complement activation, could be avoided by using mammalian antibody fragments instead of complete antibodies. For example, IgG antibodies can be enzymatically cut by papain into Fab fragments (fragment antigen binding) and Fc fragments (fragment cristallizable). Furthermore, recombinant production of Fab fragments is possible. In a preferred form, light and heavy chain domains are formed by a single peptide chain, which can be recombinantly generated (scFv, single chain fragment variable). Libraries of scFv, in particular as phage display libraries, are available in the art, which facilitate generation of recombinant antibodies or scFv specific for a given antigen. One major limitation of scFv or Fab molecules, however, is their monovalent format, impairing the affinity of these molecules and, thereby, their applicability for analytical applications. Alternatively, bivalent Fab2 fragments could be used, which still contain the hinge region, wherein the two heavy chains are connected by a disulfide linkage. Fab2 can be produced by enzymatic digestion of antibodies with pepsin. However, especially for analytical applications, another major limitation of scFv, Fab and Fab2 molecules is the lacking Fc region of the heavy chain. As a result, recognition of theses antibody fragments by secondary antibodies to the Fc region is severely impaired.
As an alternative to the above listed assay systems, basophil granulocytes have been used for the detection of allergen-specific antibodies in serum. Blood basophils together with submucosal mast cells are primary effector cells in IgE-mediated immediate-type allergic reactions such as allergic rhinitis, allergic asthma, IgE-mediated urticaria or anaphylactic shock.
The principle of the method is to challenge sensitized basophils, believed to be sensitized analogously to skin mast cells, with allergen which will cross-link surface-bound specific IgE causing histamine to be released from the cells (in vitro mediator release assay; MRA). Released histamine is determined and a dose-response curve can be constructed. In order to ensure that the basophils are responding properly, anti-IgE antibodies are applied as positive control, whereas the test substance is applied on basophils carrying no specific IgE to exclude non-specific histamine release. The histamine release tests have been shown to correlate well with other methods for in vitro measurement of allergen-specific antibodies in serum and with skin prick tests (Østergaard et al., Allergy 45: 231, 1990). More recently, the synthesis and release of leukotrienes has been applied to monitor the response of basophils upon allergen stimulation. While this assay is suitable for the detection of allergen-specific antibodies in serum, the level of free IgE in serum cannot be determined with sensitized basophils since the binding sites of their Fcε receptors are already occupied.
Basophils could be used for the determination of free IgE in serum after stripping of their original IgE by a brief treatment at low pH (Pruzansky et al., J. Immunol. 131: 1949, 1983). The stripping step, however, can interfere with the biological function of these cells, which is likely to affect the reliability of the assay. Cord blood basophils which do not require the stripping step represent an alternative, but these cells are difficult to obtain. In principle, basophil cell lines such as the KU812 (Hara et al., Biochem. Biophys. Res. Commun. 247: 542, 1998) or animal cell lines transfected with the human FcεRI (Lowe et al., J. Immunol. Methods 184: 113, 1995) may be used as recipient cells for MRAs.
However, all cell-based assay systems pose important limitations for routine analyses since they are expensive, labor intensive, and difficult to standardize.
Application of a soluble derivative of the alpha chain of human FcεRI, also referred to as FcεRIα, as capture and/or detection reagent provides another possibility for establishing an IgE assay that allows differentiation between complexed and non-complexed serum IgE. The alpha chain of FcεRI binds IgE molecules with high affinity (KD of about 10−9 to 10−10 M). Prior investigators have disclosed the nucleic acid sequence for human FcεRIα as well as for a soluble fragment thereof (U.S. Pat. No. 4,962,035, by Leder et al.; U.S. Pat. No. 5,639,660, by Kinet et al.). By introduction of a stop codon before the single C-terminal transmembrane anchor, a soluble FcεRIα fragment of 172 amino acid residues has been expressed that mediates high affinity binding of IgE (Blank et al., J Biol Chem 266, 2639-2646, 1991). The dissociation rate of bound IgE from this soluble truncated receptor was comparable to that of FcεRI on intact cells. The extracellular portion of the human FcεRIα protein contains two immunoglobulin-like domains (D1 and D2) which belong to the truncated C2 subtype of the immunoglobulin superfamily (Kinet, Annu Rev Immunol 17: 931-972, 1999). There are seven N-linked carbohydrate attachment sites in the human FcεRIα molecule. They are distributed about the front and back of the molecule, but are not found on top of the molecule (Garman et al., Annu Rev Immunol 17: 973-976, 1999). Glycosylation of FcεRIα affects the secretion and stability of the receptor, but is not required for IgE-binding (Blank et al., J Biol Chem 266: 2639-2646, 1991). However, the deglycosylated receptor has a tendency to form oligomers and aggregates in solution (Robertson, J Biol Chem 268: 12736-12743, 1993; Scarselli et al., FEBS Lett 329: 223-226, 1993; Letourner et al., J Biol Chem 270: 8249-8256, 1995). Apparently, the carbohydrates reduce the affinity of the receptor for itself, thus preventing premature aggregation on the cell surface.
The alpha chain of human FcεRI has been used for the detection of human IgE and IgE from other species including canine, feline, and equine IgE (WO 98/23964).
However, the molecular mass of the soluble truncated FcεRIα-construct (unglycosylated) is less than 20 kDa and immobilization or labelling of this small protein is likely to impair its capability as capture and detection reagent due to steric hindrance problems. IgE is a bulky ligand and high affinity binding to FcεRIα requires unrestricted interaction of all involved amino acid residues.
Steric hindrance problems can be minimized by fusion of FcεRIα With a protein suitable for immobilization and detection. Especially mammalian constant immunoglobulin domains are suitable for this purpose since a variety of established techniques are available for site-directed immobilization and detection of immunoglobulins by species-specific antibodies. The IgE binding capacity of FcεRIα is not affected by fusion to immunoglobulin domains. Using an ELISA format, chimeric molecules comprising the extracellular portion of human FcεRIα and constant domains of the heavy chain of human IgG1 have been demonstrated to bind efficiently to immobilized human IgE (Haak-Frendscho et al., J Immunol 151: 351-358, 1993). As described earlier, however, the presence of mammalian immunoglobulin domains in capture or detection reagents can lead to false positive or false negative reactions in immunological assays.
The person skilled in the art is therefore faced with the need of generating improved capture and/or detection reagents that allow differentiation between complexed and non-complexed serum IgE and avoid or minimize the above mentioned problems in the use of mammalian antibodies and fusion proteins comprising FcεRIα and constant immunoglobulin domains of mammalian origin.